Purification and Properties of 3-cis-2-trans-Enoyl-CoA Isomerase (Dodecenoyl-CoA Δ-Isomerase) from Rat Liver Mitochondria

Stoffel, Wilhelm; Grol, Michael

doi:10.1515/bchm2.1978.359.2.1777

Cited by 29 publications

(10 citation statements)

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“…Plates used to assess utilization of fatty acids were prepared by pouring a thin layer of medium at 55°C that consisted of 0.67% (w/v) yeast nitrogen base with amino acids, 0.1% (w/v) yeast extract, 0.5% (w/v) potassium phosphate at pH 6.0, and 2% (w/v) agar. The medium was autoclaved with 0.125% (w/v) palmitic acid (cis-C 16:0 ), oleic acid, cis-12-octadecenoic acid, or arachidonic acid (cis-C 20:4 (5,8,11,14) ) and with 0.5% (w/v) Tween 80 to solubilize the fatty acid used. In oleic acid liquid medium used for the same purpose, the agar was omitted.…”

Section: Methodsmentioning

confidence: 99%

Peroxisomal Δ3-cis-Δ2-trans-Enoyl-CoA Isomerase Encoded by ECI1 Is Required for Growth of the Yeast Saccharomyces cerevisiae on Unsaturated Fatty Acids

Gurvitz

Mursula

Firzinger

et al. 1998

Journal of Biological Chemistry

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We have identified the Saccharomyces cerevisiae gene ECI1 encoding ⌬ 3 -cis-⌬ 2 -trans-enoyl-CoA isomerase that acts as an auxiliary enzyme in the ␤-oxidation of (poly)-unsaturated fatty acids. A mutant devoid of Eci1p was unable to grow on media containing unsaturated fatty acids such as oleic acid but was proficient for growth when a saturated fatty acid such as palmitic acid was the sole carbon source. Levels of ECI1 transcript were elevated in cells grown on oleic acid medium due to the presence in the ECI1 promoter of an oleate response element that bound the transcription factors Pip2p and Oaf1p. Eci1p was heterologously expressed in Escherichia coli and purified to homogeneity. It was found to be a hexameric protein with a subunit of molecular mass 32,000 Da that converted 3-hexenoyl-CoA to trans-2-hexenoyl-CoA. Eci1p is the only known member of the hydratase/isomerase protein family with isomerase and/or 2-enoyl-CoA hydratase 1 activities that does not contain a conserved glutamate at its active site. Using a green fluorescent protein fusion, Eci1p was shown to be located in peroxisomes of wild-type yeast cells. Rat peroxisomal multifunctional enzyme type I containing ⌬ 3 -cis-⌬ 2 -trans-enoyl-CoA isomerase activity was expressed in ECI1-deleted yeast cells, and this restored growth on oleic acid.In mammals, degradation of fatty acids via ␤-oxidation occurs in both peroxisomes and mitochondria; however, in yeast this is solely a peroxisomal process (1). The yeast Saccharomyces cerevisiae is capable of degrading saturated fatty acids in a multistep process catalyzed by the enzymes acyl-CoA oxidase (Pox1p), 2-enoyl-CoA hydratase 2 and D-specific 3-hydroxyacylCoA dehydrogenase (Fox2p), and 3-ketoacyl-CoA thiolase (Pot1p/Fox3p) as shown in Fig. 1 (Refs. 2-5).The only double bonds of unsaturated intermediates that are catabolized via the classical ␤-oxidation pathway are at the ⌬ 2 -position and in the trans-configuration. Therefore, the removal of other double bonds requires the participation of auxiliary enzymes. Fatty acids with double bonds at odd-numbered positions such as oleic acid (cis-C 18:1(9) ) require for their degradation (along with the regular ␤-oxidation enzymes) ⌬ 3

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Section: Methodsmentioning

confidence: 99%

Peroxisomal Δ3-cis-Δ2-trans-Enoyl-CoA Isomerase Encoded by ECI1 Is Required for Growth of the Yeast Saccharomyces cerevisiae on Unsaturated Fatty Acids

Gurvitz

Mursula

Firzinger

et al. 1998

Journal of Biological Chemistry

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“…All of the characterized Δ 3 ‐Δ 2 ‐enoyl‐CoA isomerases belong to the low‐homology hydratase/isomerase protein family. Three mammalian isoforms of Δ 3 ‐Δ 2 ‐enoyl‐CoA isomerase have been identified [133–135]: two isomerases that are apparently monofunctional enzymes, and one that is an integral part of the peroxisomal multifunctional enzyme type 1, perMFE1 [135–138]. One of the two monofunctional isomerases (MECI in rat) is a predominantly mitochondrial enzyme thought to additionally occur in peroxisomes (see ) and is most active in catalyzing cis ‐3 to trans ‐2 isomerization, whereas the other dually localized monofunctional isomerase (ECI in rat) is optimal for trans ‐3 to trans ‐2 isomerization [139].…”

Section: Classical and Auxiliary Enzymes Of Peroxisomal β‐Oxidationmentioning

confidence: 99%

The biochemistry of peroxisomal β-oxidation in the yeastSaccharomyces cerevisiae

Hiltunen¹,

Mursula²,

et al. 2003

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Peroxisomal fatty acid degradation in the yeast Saccharomyces cerevisiae requires an array of beta-oxidation enzyme activities as well as a set of auxiliary activities to provide the beta-oxidation machinery with the proper substrates. The corresponding classical and auxiliary enzymes of beta-oxidation have been completely characterized, many at the structural level with the identification of catalytic residues. Import of fatty acids from the growth medium involves passive diffusion in combination with an active, protein-mediated component that includes acyl-CoA ligases, illustrating the intimate linkage between fatty acid import and activation. The main factors involved in protein import into peroxisomes are also known, but only one peroxisomal metabolite transporter has been characterized in detail, Ant1p, which exchanges intraperoxisomal AMP with cytosolic ATP. The other known transporter is Pxa1p-Pxa2p, which bears similarity to the human adrenoleukodystrophy protein ALDP. The major players in the regulation of fatty acid-induced gene expression are Pip2p and Oaf1p, which unite to form a transcription factor that binds to oleate response elements in the promoter regions of genes encoding peroxisomal proteins. Adr1p, a transcription factor, binding upstream activating sequence 1, also regulates key genes involved in beta-oxidation. The development of new, postgenomic-era tools allows for the characterization of the entire transcriptome involved in beta-oxidation and will facilitate the identification of novel proteins as well as the characterization of protein families involved in this process.

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“…The peroxisomal trifunctional enzyme (pTFE) contains an isomerase subunit, which carries out the equivalent reaction when peroxisomal ␤-oxidation is challenged, e.g. by lipid-lowering drugs (6).Mature ECI of mouse, rat, bovine, and man are 29-kDa soluble mitochondrial matrix proteins (7,8). The cDNA-derived amino acid sequence of rodents encodes a 289-residue polypeptide (32 kDa) with a 28-residue N-terminal signal sequence, and that of human encodes a 302-residue ECI (33 kDa) with a 41-residue N-terminal signal sequence, which are processed to the 261-residue mature enzyme during mitochondrial import (9 -11).…”

mentioning

confidence: 99%

Disruption of Mitochondrial β-Oxidation of Unsaturated Fatty Acids in the 3,2-trans-Enoyl-CoA Isomerase-deficient Mouse

Janßen

Stoffel

2002

Journal of Biological Chemistry

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Cellular energy metabolism is largely sustained by mitochondrial ␤-oxidation of saturated and unsaturated fatty acids. To study the role of unsaturated fatty acids in cellular lipid and energy metabolism we generated a null allelic mouse, deficient in 3,2-trans-enoyl-CoA isomerase (ECI) (eci ؊/؊ mouse). ECI is the link in mitochondrial ␤-oxidation of unsaturated and saturated fatty acids and essential for the complete degradation and for maximal energy yield. Mitochondrial ␤-oxidation of unsaturated fatty acids is interrupted in eci ؊/؊ mice at the level of their respective 3-cis-or 3-trans-enoyl-CoA intermediates. Fasting eci ؊/؊ mice accumulate unsaturated fatty acyl groups in ester lipids and deposit large amounts of triglycerides in hepatocytes (steatosis). Gene expression studies revealed the induction of peroxisome proliferator-activated receptor activation in eci ؊/؊ mice together with peroxisomal ␤-and microsomal -oxidation enzymes. Combined peroxisomal ␤-and microsomal -oxidation of the 3-enoyl-CoA intermediates leads to a specific pattern of medium chain unsaturated dicarboxylic acids excreted in the urine in high concentration (dicarboxylic aciduria). The urinary dicarboxylate pattern is a reliable diagnostic marker of the ECI genetic defect. The eci ؊/؊ mouse might be a model of a yet undefined inborn mitochondrial ␤-oxidation disorder lacking the enzyme link that channels the intermediates of unsaturated fatty acids into the ␤-oxidation spiral of saturated fatty acids.Long chain saturated and unsaturated (mono-and polyunsaturated) fatty acids comprising members of the -3 (␣-linolenic), -6 (linoleic), and -9 (oleic acid) families occur almost equally as acyl groups of phospholipids and triglycerides. In phospholipids they are essential in the regulation of the fluidity of biological membranes. -3 and -6 polyunsaturated fatty acids are the precursors in eicosanoid synthesis (prostaglandins, prostacyclins, thromboxanes, and leukotrienes). As constituents of triglycerides, unsaturated fatty acids are a main energy source for muscle work.Following the classical pathway of mitochondrial ␤-oxidation of unsaturated fatty acids with cis double bonds at odd-numbered C atoms, e.g. of oleic acid (18:1 9 ), linoleic acid (18:1 9,12 ), and ␣-linolenic acid (18:1 9,12,15 ), yields 3-cis-enoyl-CoA-intermediates. They are isomerized by the mitochondrial 3,2-transenoyl-CoA isomerase (ECI) 1 (EC 5.3.3.8) to their respective 2-trans-enoyl-CoA isomers, common substrates of enoyl-CoA hydratase of the ␤-oxidation cycle of saturated fatty acyl-CoA esters (1). cis double bonds at even C atoms yield 2-trans-4-cisintermediates, which are reduced and isomerized by a mitochondrial NADPH-dependent 2,4-dienoyl-CoA reductase (EC 1.3.1.34) to their respective 3-trans-intermediates (2-4). Hydration to the D(Ϫ)-3-hydroxy derivative followed by epimerization by 3-hydroxyacyl-CoA epimerase (EC 5.1.2.3) is apparently an alternative but minor pathway. Likewise, another alternative pathway has been proposed, according to which a cis-5 do...

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Purification and Properties of 3-cis-2-trans-Enoyl-CoA Isomerase (Dodecenoyl-CoA Δ-Isomerase) from Rat Liver Mitochondria

Cited by 29 publications

References 3 publications

Peroxisomal Δ3-cis-Δ2-trans-Enoyl-CoA Isomerase Encoded by ECI1 Is Required for Growth of the Yeast Saccharomyces cerevisiae on Unsaturated Fatty Acids

Peroxisomal Δ3-cis-Δ2-trans-Enoyl-CoA Isomerase Encoded by ECI1 Is Required for Growth of the Yeast Saccharomyces cerevisiae on Unsaturated Fatty Acids

The biochemistry of peroxisomal β-oxidation in the yeastSaccharomyces cerevisiae

Disruption of Mitochondrial β-Oxidation of Unsaturated Fatty Acids in the 3,2-trans-Enoyl-CoA Isomerase-deficient Mouse

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